1. Field of the Invention
The present invention relates to a polyhydroxyalkanoates (PHAs) production method. More than 95% of the PHAs are comprised of poly(3-hydroxyvalerate-co-4-hydroxyvalerate) (P3HV-co-P4HV). PHAs are produced by Bacillus sp., which is cultivated under the condition with unbalanced nutrients and supplemented with a carbon source of succinate.
2. The Prior Arts
Plastic materials produced from traditional petrochemical industry are light weight and durable, and are convenient for daily use. However, they increasingly contribute to serious environmental problems such as pollution because of their persistence. Additionally, the limited availability and high price of crude oil make it a very expensive starting material for PHA production. Therefore, the development of a sustainable substitute source for plastic production is urgently needed. Additionally, this new family of plastic will be biodegradable. Currently there are several types of biodegradable materials:
(1) Photodegradable Plastics
There have been numerous studies done in photodegradable plastics than in other degradable plastics. These products are created by introducing photoactive additives into copolymers which can be degraded when exposed to ultraviolet light. However, secondary pollution can be caused due to incomplete degradation of these plastics.
(2) Compostable Plastics
These plastics are made from natural resources like corn protein or plant starch added in plastic materials. However, they have poor resistance to water and heat rendering their daily use impractical.
(3) Chemical Synthesized Degradable Plastics
These products are synthesized from direct polymerization techniques, using polylactides, that are prepared by direct polycondensation of lactic acid. Polylactides are biodegradable and biocompatible but their molecular weights are low, and production costs are higher than those of the petrochemical materials.
(4) Polyhydroxyalkanotes (PHAs)
PHAs are polyesters produced by microorganisms. Hydroxyalkanoic acid are accumulated and converted to PHAs in microorganisms when they are grown under unbalanced nutritional conditions, for example, limiting in nitrogen source or other minimal elements (potassium, iron, sulfur, phosphate and so on), while a carbon source is provided in excess. Polyesters are stored in the microorganisms as an emergency energy source when carbon source from the environment is used up. Stored PHAs can be degraded into carbon dioxide and water by intracellular enzymes therefore generating energy for use.
The physical properties of PHAs are similar to those of petrochemical plastics such as polyethylene. Therefore they can be good alternatives to petrochemical plastics because of their biodegradability. Their production and use will alleviate problems caused by high cost of petroleum and its contribution to pollution. Moreover, the merits of good biocompatibility, biodegradability, and flexibility make PHAs good research candidates for biomedical materials with high values to be applied in bioengineering and biomedical industry.
Over 150 species of microorganisms, including Gram-positive, Gram-negative bacteria from different Genera, and some Archaea were found to produce PHAs.
The general structure of PHAs is shown below. The side chain R can be alkanes with different number of carbon atoms. Different kinds of PHAs are formed with different alkanes. For example, short-chain-length PHAs (scl-PHAs) are composed of monomers containing 3-5 carbon atoms. Poly(3-hydroxybutyrate) [P(3HB) or PHB] in this group contains monomers of 3-hydroxybutyrate (3HB); and Poly(hydroxybutyrate-hydroxyvalerate) (PHBV) is a copolymer comprised of monomers 3HB and 3-hydroxyvalerate (3HV). Medium-chain-length PHA (mcl-PHA) is composed of monomers containing 6-10 carbon atoms.

n=1, R=hydrogen, PHAs=poly(3-hyroxypropionate), P(3HP)                R=methyl, PHAs=poly(3-hydroxybutyrate), P(3HB)        R=ethyl, PHAs=poly(3-hydroxyvalerate), P(3HV)        R=propyl, PHAs=poly(3-hydroxycaproate), P(3HC)        R=butyl, PHAs=poly(3-hydroxyheptanoate), P(3HH)        R=pentyl, PHAs=poly(3-hydroxyocatanoate), P(3HO)        R=hexyl, PHAs=poly(3-hydroxynonanoate), P(3HN)        R=heptyl, PHAs=poly(3-hydroxydecanoate), P(3HD)        R=octyl, PHAs=poly(3-hydroxyundecanoate), P(3HUD)        R=nonyl, PHAs=poly(3-hydroxydodecanoate), P(3HDD)        
n=2, R=hydrogen, PHAs=poly(4-hydroxybutyrate), P(4HB)                R=methyl, PHAs=poly(4-hydroxyvalerate), P(4HV)        
n=3, R=hydrogen, PHAs=poly(5-hydroxyvalerate), P(5HV)
The basic structure of PHAs consists mainly of monomeric units of hydroxyalkanoates (HA). The hydroxyl group of one monomer is attached to the carboxyl group of another by an ester bond to form a long chain type polyester accumulation. The alkyl group (R) in the C-3 or β position of the monomer can be groups of saturated, unsaturated, alcohol or alkanoate with side chain. PHAs are classified according to the type of alkyl group in the β position.
A variety of metabolic pathways and enzymes can synthesize PHAs with different compositions. Different bacteria have different metabolic pathways and enzymes involved in PHA production, which results in the preferential production of a specific type of PHA. Three metabolic pathways for PHA production in microorganisms are found: glycolysis as shown in FIG. 1A, fatty acids degradation as shown in FIG. 1B, and fatty acids biosynthesis as shown in FIG. 1C.
As illustrated in FIG. 1A, acetyl-CoA produced from glycolysis of glucose is converted to acetoacetyl-CoA through catalysis of β-ketothiolase. Acetoacetyl-CoA is reduced by acetoacetyl-CoA reductase to form (R)-3-Hydroxybutyryl-CoA. Short chain length PHAs (scl-PHAs) are produced in the last step through polymerization of PHB polymerase.
As illustrated in FIG. 1B, microorganisms such as Aeromonas sp. or Pseudomonas sp. use fatty acids as carbon source. Fatty acids can be degraded into short chain fatty acid monomers through β-oxidation and form intermediate products, which include acyl-CoA, enoyl-CoA, (S)-3-hydroxyacyl-CoA, and 3-ketoacyl-CoA. Middle products of enoyl-CoA, (S)-3-hydroxyacyl-CoA, and 3-ketoacyl-CoA which can be catalyzed into (R)-3-hydroxyacyl-CoA through the function of enoyl-CoA hydratase, epimerase and ketoacyl-CoA reductase respectively. In the last step, medium or long chain length PHAs (mcl-PHAs or lcl-PHAs) are formed through the function of PHA synthases (PhaC).
As illustrated in FIG. 1C, different carbon sources could be degraded by microorganisms to yield acetyl-CoA. The acetyl-CoA would not be used to directly produce scl-PHAs but synthesize fatty acids. The intermediate R-3-hydroxyacyl-ACP is converted to R-3-hydroxyacyl-CoA and mcl-PHAs through the catalysis of PhaG (CoA transacylase) and PhaC accordingly.
The important cost factor for the commercialization of microbially-synthesized PHAs is the selection of carbon source for microbial growth substrates. The high cost of traditional culture methods makes PHAs difficult to compete with petrochemical plastics. However, unlike most petrochemical plastics, PHAs is biocompatible and biodegradable. And more than 150 types of PHAs were discovered so far. These properties of PHAs are good for biomedical materials with high values to be applied in bioengineering and biomedical industry. Different PHAs can be produced through different carbon sources or growth substrates accompanied with different strains.
Bacillus megaterium belongs to the Bacillus sp., which was discovered first to synthesize PHAs and was cultivated in a variety of carbon sources. Attempts at cultivation with different carbon sources were carried out to lower the production cost of PHAs or to produce special PHAs with high values.
The inventor of the invention has discovered a production method for polyhydroxyalkanoates (PHAs) with majority of poly(3-hydroxyvalerate-co-4-hydroxyvalerate) (P3HV-co-P4HV) using succinate as a carbon source after testing several possible carbon sources.